title: B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.
abstract: ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.
authors.affiliation: Institute of Immunology and Rheumatology, University of Oslo, Norway. morten.harboe@labmed.uio.no
authors.forename: M
authors.initials: M
authors.lastname: Harboe
authors.forename: A S
authors.initials: AS
authors.lastname: Malin
authors.forename: H S
authors.initials: HS
authors.lastname: Dockrell
authors.forename: H G
authors.initials: HG
authors.lastname: Wiker
authors.forename: G
authors.initials: G
authors.lastname: Ulvund
authors.forename: A
authors.initials: A
authors.lastname: Holm
authors.forename: M C
authors.initials: MC
authors.lastname: Jørgensen
authors.forename: P
authors.initials: P
authors.lastname: Andersen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis / Q000276:immunology*; D001426:Bacterial Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D018985:Epitopes, B-Lymphocyte